13C glucose breath test for the diagnosis of diabetic indications and monitoring glycemic control

ABSTRACT

A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired  13 C-labeled CO 2  following the ingestion of a  13 C-enriched glucose source.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/674,806, filed Feb. 8, 2001, now U.S. Pat. No. 6,468,802, which is aU.S. National Phase Application of International ApplicationPCT/IB99/00933, filed May 6, 1999, which claims priority to U.S.Provisional Application No. 60/084,482, filed May 6, 1998. Thedisclosure of each of the above applications is incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Glucose tolerance is defined as the ability to properly utilize glucose.Diabetes is not a single disease, but an array of diseases that exhibitthe common symptom of glucose intolerance, an impairment in glucoseutilization.

The prevalence of diabetes in the general population is approximately6-7%. Only about half of diabetics are actually diagnosed. Studies haveshown that rates for persons with glucose intolerance are equal by sexand greater for blacks than for whites.

In general, the following types of diabetes have been recognized: type Idiabetes mellitus, type II diabetes mellitus, secondary diabetesmellitus, impaired glucose tolerance and gestational diabetes mellitus.The general characteristics of the symptoms of diabetes include thefollowing:

Polyuria (excretion of large quantities of urine)

Hyperglycemia (high blood glucose levels)

Glucosuria ( abnormal presence of glucose in urine)

Polydipsia (excessive thirst)

Polyphagia (excessive hunger)

Sudden weight loss.

It has been observed that complications resulting from diabetes mellitusare the third leading cause of death in most developed countries.Diabetes is a risk factor for a variety of conditions including coronaryheart disease, cerebrovascular stroke, neuropathy (nerve damage),nephropathy (kidney damage), retinopathy (eye damage), hyperlipidemia(excessive blood lipids), angiopathy (damage to blood vessels) andinfection.

A number of different methods exist for determining a condition ofintolerance for glucose. These include postprandial blood glucose, oralglucose tolerance test (OGTT), O'Sullivan glucose tolerance test(gestational test), hemoglobin Alc (Hb A₁, Hb A_(1c)), islet cellantibodies, GAD antibodies (glutamic acid decarboxylase) and insulinantibodies. Diabetes, however, is most readily detected when thecarbohydrate metabolic capacity is tested. This is done by stressing thesystem with a defined glucose load as in the oral glucose tolerance test(OGTT).

The OGTT has been criticized, however, because many of the variablesaffecting test results are difficult to control, for instance: Patientsmust be on a standardized carbohydrate diet at least three days beforethe test. The test requires an 8 to 16 hour fast. The test should onlybe performed on ambulatory patients. Stress should be avoided. Exerciseshould be avoided. Various hormone imbalances can affect validity suchas with: thyroxine, growth hormone, cortisol and catecholamines. Variousdrugs and medications can affect validity such as: oral contraceptives,salicylates, nicotinic acid, diuretics and hypoglycemics. Evaluationshould normally be corrected for age. The greatest disadvantage of theOGTT is that it is poorly reproducible and this limits its diagnosticusefulness.

The current methods of diagnosing diabetes involve either invasivetesting (ie. repeated blood collections), or use blood-borne markers(ie. glycosylated proteins, or antibodies) which offer an indirectassessment of glucose regulation. Accordingly, it is an object of thepresent invention to avoid the need for invasive testing or the use ofblood-borne markers in determinations of glucose regulation.

SUMMARY OF THE INVENTION

The above and other objects of the invention are attained by a ¹³Cbreath test and a kit for determining glucose regulation in a patient inneed thereof.

An analytical assay is described that is based on the use ofnon-radioactive ¹³C. Labeled expired ¹³CO₂ is measured in the presentassay. Isotope ratio mass spectroscopy (IRMS) is used as a detectionmethod for ¹³C, a non-radioactive isotope that occurs naturally in foodand animal tissues. Non-dispersive infrared spectroscopy (NDIRS)analysis and analysis methods known in the art may be employed. The testprotocol is as follows: after an overnight fast, the oral dose of ¹³Cuniformly labeled glucose (containing about 25 mg of ¹³C glucose incombination with about 15 g of unlabeled glucose in 100 mL of tap water)is administered. Breath samples will be collected before the dose andthen 1½ hours after ¹³C glucose ingestion. Levels of ¹³CO₂ in expiredair will be measured by an IRMS method.

Advantages of this test are the following:

it is practical, sensitive and specific;

the validity of the test is not influenced by stress, exercise, hormoneimbalances, or some drugs and medications

it is a non-invasive method;

it is simple to perform and can be readily used in physicians' officesor medical laboratories;

it is safe since ¹³C is a naturally occurring isotope found in allcarbon-containing substances;

it involves no radioactivity, and may be used in children and women.

The ¹³C glucose test is safe, reliable, and specific in diagnosis ofdiabetes and measurement of the severity of insulin resistance inpatients. The invention is also preferred to diagnose gestationaldiabetes and to monitor glycemic control in diabetes patients. Apreferred embodiment of the invention is a kit containing the necessarymaterial for performing the described method. This kit may contain butis not limited to a source of ¹³C enriched glucose (preferably uniformlylabeled D-glucose); a source of unenriched glucose; and a breathcollection device. The kit may also contain a set of patientinstructions for its use. In another embodiment, the kit mayadditionally contain a blood collection device such as a lancet orhypodermic needle and vacutainer for the additional determination ofblood glucose levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Illustrates the IRMS analysis of ¹³C glucose breath samples fromnormal individuals, a gestational diabetic and patients with impairedglucose tolerance.

FIG. 2: Shows a representative example of breath test and blood glucoselevels of a normal individual.

FIG. 3: Illustrates breath test and blood glucose levels of a diabeticpatient.

FIG. 4: Depicts breath test and blood glucose levels of an insulinresistant patient.

FIG. 5: Shows a comparison of IRMS results of an insulin resistant and adiabetic patient and a normal individual.

DETAILED DESCRIPTION OF THE INVENTION

The introduction of a ¹³C breath test offers a novel, non-invasive,direct means to monitor glucose metabolism by measurement of exhaled CO₂using highly enriched, uniformly labeled ¹³C-glucose. Glucose metabolismwill generate labeled CO₂, which is then exhaled and collected in tubes.Enrichment of labeled CO₂, over a determined time course, can be used asa quantitative index of glucose metabolism. Comparison is made againstage-specific reference intervals.

The present invention has a number of advantages, including lower doseof glucose needed (overcomes inconsistencies due to malabsorptivedisorders or previous gastric or intestinal surgery), reduction intesting time (from the current 2 hours required for the OGTT) and fewerinterpretational ambiguities (greater sensitivity and specificity).

The ¹³C glucose breath test is based on the metabolism of glucose.Following a baseline breath sample, a ¹³C glucose solution containingabout 25 mg of ¹³C glucose in combination with about 15 g of unlabeledglucose in 100 mL of tap water is administered. Breath samples will beobtained before the dose and then 12 hours after ¹³C glucose ingestion.Measurement of the expired air will be detected by an isotope ratio massspectroscopy assay method. Elevated or excessive breath 13CO2concentrations will be seen in individuals who have normal glucosemetabolism.

The following Examples serve to illustrate the present invention. TheseExamples are not intended to limit the scope of the invention in anymanner.

EXAMPLE 1 Sample Assay for Diagnosis of a Patient

Experimental Procedure

Medical History

Medical history is taken and includes, but is not limited to: theabsence of active pulmonary disease, no history of heart, liver, orrenal failure, and no use of insulin or oral medications for thetreatment of diabetes.

Physical Examination and Laboratory Tests

No physical examination or laboratory tests, including blood sampling,is required.

Dietary Control

It is determined that all participants have fasted overnight prior tocommencement of the test.

Patient Control

Participants are not permitted to eat, drink, or smoke during the test.All patients are required to remain sedentary for the duration of thetest. Small amounts of water are allowed.

Assay Procedure

Patients fast for at least 8 hours before this test.

A sample set of patient instructions is given below:

Step 1: COLLECT FIRST BREATH SAMPLE

Remove the screw cap from the collection tube.

Take a normal breath and then exhale fully 4 to 8 seconds through astraw into the bottom of the collection tube.

Immediately replace the screw cap on the collection tube and tightenuntil snug (do not overtighten).

Affix the completed green label to the collection tube.

Step 2: DRINK THE SOLUTION

Prepare the solution by adding tap water to the fill line on the plasticcontainer. Mix until completely dissolved and then drink the entiresolution.

Wait 1½ hours.

Step 3: COLLECT THE SECOND BREATH SAMPLE

One and one half hours after drinking the solution, collect the secondbreath sample into the collection tube following the same directions asfor the first breath sample in step 1.

Affix the completed yellow label to the tube.

Step 4: RETURN THE SAMPLES FOR ANALYSIS

Insert the 2 collection tubes along with the signed and completedregistration card in the mailing box.

Return the mailing box as instructed to the site of dispensing.

EXAMPLE 2 Breath Test Administration

Patients are given an exetainer tube with the screw cap removed. Usingthe straw, they are asked to breathe into the tube, exhaling normally,for 4 to 8 seconds. Next, each patient is instructed to drink a solutioncontaining about 25 mg of uniformly labeled ¹³C glucose in combinationwith about 15 g of unlabeled glucose in 100 mL of tap water. After 12hours, the patients are given a new tube to breathe in as describedabove. The breath collection is then complete.

Storage and Shipping

Breath test tubes are typically labeled with the patient's name andidentification number and shipped to an analytical laboratory foranalysis. No refrigeration or special storage techniques are necessary.

EXAMPLE 3 Analytical Methodology

Breath specimens are analyzed by isotope ratio mass spectroscopy. NDIRSis also a preferred method to analyze breath test samples. Other methodsknown in the art may also be used.

Statistical Analysis

The sensitivity, specificity, positive and negative predictive values ofthe breath test are compared to that of the oral glucose tolerance test.Receiver operator characteristic curve analysis is performed to confirmthe discrimination between type 2 diabetes or gestational diabetes andindividuals with normal glucose metabolism.

EXAMPLE 4 Basis of the Method of IRMS

Isotope ratio mass spectroscopy (IRMS) is a highly precise method ofanalysis which is able to measure small samples (low nanogram amounts).For example, ¹³C/¹²C ratios are determined on a mono-carbon molecule;CO₂ gas. The CO₂ gas can be directed to the spectrometer by means of acontinuous flow IRMS (also called CF-IRMS).

The statistical combination of the isotopes of carbon (¹²C and ¹³C) andoxygen (¹⁶O, ¹⁷O, ¹⁸O) to generate the CO₂ molecules gives rise to theformation of various isotopomers whose molecular weights are 44, 45, and46, respectively. Thus, for measuring carbon isotope ratios, three ionbeams are generated and recorded in the IRMS, corresponding to themasses of the various isotopomers of CO₂.

In order to obtain a high precision and a high accuracy, reference gasesof absolutely known isotopic composition are used and a dual inletsystem allows an alternative admission of both sample and referencegases into the ionization source via a gas-switching valve. Themeasurement of the various ion beams allows for the calculation of the¹³C enrichment of the sample. The value of this calculation is givenδ¹³C(%) notation. The ¹³C abundance is expressed as δ¹³C(%) according tothe following:

δ¹³C(%)=([(¹³C/¹²C) sample/(¹³C/¹²C)PDB]−1)×1000

This δ¹³C(%) value measures the variations in parts per thousand of thecarbon isotope ratio from the standard. For carbon, PDB was selected asthe international reference. PDB is Pee Dee Belemnitella (a fossil fromthe Pee Dee geological formation in South Carolina). The ¹³C/¹²C ratiofrom the calcium carbonate of this fossil is 0.011237. Compared to PDB,most of the natural compounds display a negative delta value. In theabove equation, ¹³C/¹²C refers to the isotopomers.

Using the breath test of this invention, IRMS is an example method todiagnose type 2 and gestational diabetes, and for monitoring glycemiccontrol of diabetes patients.

EXAMPLE 5 ¹³C Glucose Breath Test Results of Normal, GestationalDiabetes and Impaired Glucose Tolerance Patient

Example 4 describes a method to analyze breath samples of thisinvention. FIG. 1 shows the mean (±SD) Delta per mil over Baseline (DOB)of the normal population. Also shown are the DOB's of a gestationaldiabetic and impaired glucose tolerance patients. Breath samplescollected 0, 1, 1.5 and 2 hours according to the protocol were analyzedby IRMS. IRMS analysis of the collected breath samples can be performedon various instruments, including but not limited to the AP2003 andAP2002 (Analytical Precision Ltd), ABCA (POZ Europa) and the Breath MAT(Finnigan MAT). The DOB values of the gestational diabetes and theimpaired glucose tolerance patients are well below the DOB of the normalpopulation (FIG. 1). The impaired glucose tolerance diagnosis wasinitially determined by OGTT, the gestational diabetes screen was usedto confirm gestational diabetes.

Impaired glucose tolerance (IGT) refers to a condition in which bloodsugar levels are higher than normal, but are not high enough to beclassified as diabetes. IGT is a major risk factor for type 2 diabetes.IGT is present in about 11 percent of adults, or approximately 20million Americans. About 40-45 percent of persons age 65 years of age orolder have either type 2 diabetes or IGT. A person is currentlydiagnosed with IGT when the 2-hour glucose results from a glucosetolerance test are greater than 7.8 mmol/L, but less than 11.0 mmol/L. Awoman is diagnosed with gestational diabetes when she is pregnant andhas any two of the following: a fasting plasma glucose of more than 5.3mmol/L, a 1-hour glucose level of more than 10.6 mmol/L, a 2-hourglucose level of more than 8.9 mmol/L. However, as this method ofdiagnosis is invasive, the breath tests of the current invention is thepreferred diagnosis method. The ¹³C glucose breath test is sensitive,accurate and non-invasive.

EXAMPLE 6 ¹³C Glucose Breath Test Results of a Normal, Insulin Resistantand Diabetes Patient

In this example, both breath test and blood glucose levels were done ona normal, diabetic and insulin resistant patient. FIG. 2 shows the DOBof 0, 1, 1.5 and 2 hours breath samples of a normal subject analyzed byIRMS. The blood glucose level of this normal individual is alsodisplayed.

FIG. 3 illustrates the breath test and blood glucose levels of adiabetic patient. The DOB of the breath samples are significantly lowerthan the DOB of the normal individual (FIG. 2), the blood glucose levelsare typical of a diabetic patient.

In FIG. 4, the breath test and blood glucose levels of aninsulin-resistant patient are depicted. The DOB of these breath samplesare significantly lower than the normal DOB (FIG. 2), the blood glucoselevels are typical of an insulin-resistant patient.

These results demonstrate one preferred utility of the breath test ofthe current invention to diagnose diabetes and insulin resistance. Inanother aspect of the invention, the areas between the breath test andblood glucose test curves can be used to diagnose patients with insulinresistant or diabetes and confirm glucose tolerance in normalindividuals by the comparison of the areas to the different groups ofnormal, diabetic and insulin resistant patients.

FIG. 5 illustrates the ¹³C glucose breath test results of a normalindividual, insulin resistant and diabetes patient. The DOB's of theinsulin resistant and diabetes patients is significantly lower than thatof the normal DOB results.

EXAMPLE 7 NDIRS Instrumentation

Breath test samples of the invention can also be analyzed using NDIRSinstrumentation. The course of the ¹³CO₂/¹²CO₂ ratio in breath allowsfor diagnosis of diabetes. NDIRS can be further used to diagnose type 2and gestational diabetes patients and for monitoring therapy of diabetespatients (glycemic control of these patients).

The metabolism of ¹³C labeled substrate leads to a different isotoperatio. NDIRS analysis of the invention can be performed on variousinstruments, including but not limited to the MicroLyzer (QuinTron),UbiT-IR200 and UbiT-100 (Otsuka Pharmaceutical Co., Ltd.), the URAS 10(Hartmann and Braun) and the Isomax 2000 (Isotechnika).

EXAMPLE 8 Hyperinsulinemic Euglycemic Clamp Method for the Measurementof Insulin Resistance

Insulin resistance is defined as the decrease of the biological actionof insulin, and it mainly presents as an hyperinsulinemia. Thehyperinsulinemic euglycemic clamp is currently the reference method forquantifying insulin resistance. The clamp technique consists of infusinginsulin at a constant rate and, to prevent any decrease in the plasmaglucose level, by infusing dextrose. The rate of dextrose infused tomaintain euglycemia is an estimate of the amount of glucose, which istaken up by the tissues under the effect of a defined plasma insulinconcentration. Using several rates of insulin infusion allows theestablishment of the relationship between the whole body glucosedisposal and plasma insulin levels, and to discriminate between thestates of decreased insulin sensitivity and/or altered maximal capacityto dispose of glucose. However, the hyperinsulinemic euglycemic clampmethod is very invasive, time consuming, costly and variable. The breathtest of this invention is a preferred method to measure insulinresistance as it is reliable, sensitive, specific, cost-effective andnon-invasive.

EXAMPLE 9 Monitoring Long-Term Control of Diabetes

Measuring glycated hemoglobin is a current test used for monitoringlong-term control of diabetes. Glycated hemoglobins are increased as areflection of hyperglycemia during the lifespan of erythrocytes.However, different analytical methods may measure different glycatedhemoglobins and caution must be exercised in the interpretation ofresults. HPLC or column chromatography methods used to analyse glycatedhemoglobin are also highly sensitive to variations in temperature andpH. This test is also invasive, requiring several blood samples. Thebreath test of the present invention is preferred as it is non-invasive,sensitive, accurate and cost-effective.

EXAMPLE 10 Usefulness of ¹³C Glucose Breath Test in Diagnosis ofDiabetes

Diabetes mellitus is a group of diseases characterized by high levels ofblood glucose resulting from defects in insulin secretion, insulinaction, or both. Diabetes can be associated with serious complicationsand premature death if left undiagnosed and untreated. It has beenestimated by the World Health Organization that the number of peoplesuffering from diabetes worldwide will more than double from about 135million now to 300 million by the year 2025. Of those estimated to havediabetes, it is believed that approximately one third of those areundiagnosed. It is also known that the prevalence of diabetes increaseswith age. It is estimated that 0.16% of people under the age of 20 havediabetes but this number dramatically increases to 18.4% for people overthe age of 65.

There are four types of diabetes; type 1 (insulin dependent) represents5 to 10% of all diagnosed cases, type 2 (non-insulin-dependent diabetes)represents 90 to 95% of all diagnosed cases, gestational diabetesdevelops in 2 to 5% of all pregnancies but disappears when a pregnancyis over, and other specific types of diabetes resulting from specificgenetic syndromes, surgery, drugs, malnutrition, infections and otherillnesses may account for 1 to 2% of all diagnosed cases. A number ofdifferent methods exist for determining diabetes. These includepostprandial blood glucose, oral glucose tolerance test (OGTT),O'Sullivan glucose tolerance test (gestational test), hemoglobin Alc,islet cell antibodies, glutamic acid decarboxylase (GAD) antibodies, andinsulin antibodies. However, diabetes is most readily detected when thecarbohydrate metabolic capacity is tested. This is done by stressing thesystem with a defined glucose load as in the OGTT.

Although the OGTT is a standard test for diabetes, it has beencriticized because many of the variables affecting the test results aredifficult to control for; the standardized carbohydrate diet, eight tosixteen hour fast, stress, exercise, hormone imbalances, and variousdrugs can cause test variables. These variables lead to poorreproducibility and limit the diagnostic usefulness of this test. Inaddition, the OGTT involves the collection of numerous blood specimensmaking it an invasive procedure.

The development of a ¹³C-glucose breath test for the detection ofdiabetes offers a non-invasive method that is not affected by the abovementioned variables. ¹³C is a non-radioactive isotope that occursnaturally in food and animal tissues. In the past the disadvantage of¹³C had been the shortage of the gas isotope mass spectrometers used foranalysis. With the ready availability of the necessary instrumentationand the ¹³C-labeled compounds required, the use of ¹³C-labeled compoundsin breath tests is more feasible.

Clinical Study

Objective: The primary aim of this pilot study is to evaluate thesensitivity, specificity and reliability of a ¹³C-D-glucose breath testin the diagnosis of type 2 and gestational diabetes as compared to thealready validated glucose tolerance test that will be considered thestandard.

Design: A multi-center, blinded, non-randomized design is utilized. Onlythe referring physicians have knowledge of the participants' status.Participants undergo a glucose tolerance test. Within two weeksfollowing, participants undergo a ¹³C-D-glucose breath test. Thefindings from both tests are examined for concordance.

STUDY PARTICIPANTS: This investigation is carried out by recruiting 50individuals each for type 2 and gestational diabetes. For type 2diabetes, the participants are suspected to be diabetic. For gestationaldiabetes, the participants are women in their 24th to 28th week ofpregnancy who have presented for the standard gestational diabetesmellitus screening test. Any diagnosis of diabetes is based on theresults of the glucose tolerance test.

TESTING STRATEGY: Eligible participants, after giving informed consent,undergo the glucose tolerance test and the 13C-D-glucose breath testseparated by a minimum of 24 hours and a maximum of two weeks. Theglucose tolerance test is performed according to the guidelines of theCanadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly, for the gestational diabetes screen, theglucose tolerance test consists of the consumption of a 50 g glucosetolerance drink and the collection of a venous blood sample one hourlater for glucose determination. For the time between the drinkconsumption and the blood sampling, the participant remains sedentaryand refrains from smoking or eating. Small sips of water may be taken ifnecessary.

For type 2 diabetes, an overnight fast (10-16 hours) precedes theglucose tolerance test. A fasting glucose blood sample is drawn prior tothe consumption of a 75 g glucose tolerance drink. Two hours after theingestion of the drink, a venous blood sample is collected for glucosedetermination. For the time between the drink consumption and the bloodsampling, the participant remains sedentary and refrains from smoking oreating. Small sips of water may be taken if necessary.

The ¹³C-D-glucose breath test is preceded by an overnight fast (minimumeight hours). After fasting, the participants are required to provide abaseline breath sample. The participants then ingest the ¹³C-D-glucosedrink preparation and will provide breath samples at 1, 1.5, and 2hours. During the test the participants remain sedentary and are notpermitted to smoke or eat. Only small sips of water are permitted duringthe test.

OVERALL STUDY DESIGN: A total of 50 participants are investigated eachfor type 2 and gestational diabetes.

Visit One: During the recruitment process, each individual is asked toreview a Participant Information Sheet and to talk with the laboratorypersonnel to ensure that all eligibility requirements are met. Theindividual is given an opportunity to ask questions and if they meet allthe eligibility criteria, they are asked to read and sign an informedConsent Form.

All participants who have met the eligibility criteria and signed aconsent form are tested by both the glucose tolerance test (Visit Two)and ¹³C-D-glucose breath test (Visit Three) separated by a minimum of 24hours and a maximum of two weeks.

Visit Two: The glucose tolerance test follows the guidelines set out bythe Canadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly, for the gestational diabetes screen, theparticipants are asked to consume a commercially available glucosetolerance drink consisting of 50 g of dextrose in 296 mL. One hourfollowing consumption, a venous blood sample is collected into ared-topped vacutainer tube. For type 2 diabetes, participants firstcomplete an overnight fast (10-16 hours) and then provide a fastingblood glucose sample. Participants then ingest a commercially availableglucose tolerance drink consisting of 75 g of dextrose in 296 mLfollowed by the collection of a venous blood sample 2 hourspost-consumption.

Visit Three: For the ¹³C-D-glucose breath test, participants firstcomplete an overnight fast (minimum of 8 hours). Participants provide abaseline breath sample which is followed by consumption of a¹³C-D-glucose-enriched solution containing 25 mg of ¹³C-D-glucose incombination with 15 g of unlabeled USP dextrose in 100 ml of water.Participants then provide breath samples at 1, 1.5, and 2 hours.

Note: Visit One and Visit Two may be combined if it is more convenientand all the testing criteria are met.

NUMBER OF PARTICIPANTS AND TARGET POPULATION: A total of 100 adultparticipants (18 years of age or older) who are suspected of having type2 diabetes (n=50) or are being screened for gestational diabetes (n=50)are recruited from those individuals presenting for the oral glucosetolerance test.

INTERIM ANALYSIS: After 25 participants are enrolled for a particulartype of diabetes, all parties are unblinded to the participants' status.At this point in the study, the results are evaluated. If the¹³C-D-glucose breath test results do not correlate with the standard,the oral glucose tolerance test, such that greater than 5% of theparticipants are reported as false negatives or false positives, thestudy is temporarily halted. If the study is halted, the protocol isamended to reflect an adjustment in the ¹³C-D-glucose breath test kitcomponents such that it contains 50 mg of ¹³C-D-glucose and 15 g ofunlabeled USP dextrose.

EXAMPLE 11 Advantages of the ¹³C Glucose Test for the Diagnosis ofDiabetes

The disadvantages of the OGTT include uncontrollable factors which causevariability or spurious results and the invasiveness of the test. Othertests known in the art are not specific, are invasive, are variable andare labor intensive. The ¹³C glucose breath test of the presentinvention is sensitive, reliable and specific. The ¹³C glucose breathtest shows minimal intra-individual variation, excellent analyticalprecision and breath specimens are stable for at least six weeks at roomtemperature. The ¹³C glucose breath test is preferred over tests knownin the art, it is non-invasive, easy to perform, has very goodsensitivity and specificity and is cost effective. A preferred use ofthe breath test of this invention is for the diagnosis of type 2 andgestational diabetes. This invention is also preferred to determine thelevel of insulin resistance and for monitoring the appropriateness ofthe therapy of diabetes patients.

Further variations and modification of the present invention will beapparent to those skilled in the art and are intended to be encompassedby the specification and claims appended hereto.

We claim:
 1. A diagnostic kit for the determination of baseline glucosemetabolism in the absence of an indication of glucose intolerance,diabetes or gestational diabetes in a subject comprising: apredetermined quantity of uniformly labeled ¹³C-enriched glucose; and abreath collection container.
 2. A diagnostic kit according to claim 1,said diagnostic kit comprising at least two breath collectioncontainers.
 3. A diagnostic kit according to claim 1, said diagnostickit comprising a first breath collection container for receiving abaseline breath sample taken from the subject prior to ingestion of anythe predetermined quantity of the uniformly labeled ¹³C-enrichedglucose; and a second breath collection container for receiving a breathsample after the uniformly labeled ¹³C-enriched glucose is ingested. 4.A diagnostic kit according to claim 3, said diagnostic kit furthercomprising a set of instructions wherein the instructions direct thesubject to collect a first breath sample in said first breath collectioncontainer, ingest the uniformly labeled ¹³C-enriched glucose and collecta second breath sample at a time point that is after ingestion of theuniformly labeled ¹³C-enriched glucose, in said second breath collectioncontainer.
 5. A diagnostic kit according to claim 1, said diagnostic kitfurther comprising a tube that transfers the breath of the subject intothe breath collection container.
 6. A diagnostic kit according to claim1, said diagnostic kit further including analysis means for measuring¹³CO₂ metabolized from said uniformly labeled ¹³C-enriched glucose, in abreath sample, said analysis means being selected from the groupconsisting of an isotope ratio mass spectroscope (IRMS), a continuousflow isotope ratio mass spectroscope (CF-IRMS) and a non-dispersiveinfrared spectroscopy (NDIRS).
 7. The diagnostic kit of claim 6 whereinsaid analysis means is CF-IRMS.
 8. The diagnostic kit of claim 6 whereinsaid analysis means is NDIRS.
 9. A diagnostic kit according to claim 1,wherein said predetermined quantity contains 25 mg or less of theuniformly labeled ¹³C-enriched glucose.
 10. A diagnostic kit accordingto claim 1, further comprising about 15 g of unlabeled glucose.
 11. Adiagnostic kit according to claim 1, wherein said predetermined quantitycomprises about 25 mg of the uniformly labeled ¹³C-enriched glucose. 12.A diagnostic kit according to claim 11, said diagnostic kit comprising abreath collection container for receiving a baseline breath sample takenfrom the subject prior to ingestion of any of the predetermined quantityof the uniformly labeled ¹³C-enriched glucose; and at least two breathcollection containers for receiving a breath sample at predeterminedtime points after ingestion of the uniformly labeled ¹³C-enrichedglucose.
 13. A diagnostic kit according to claim 11, said diagnostic kitcomprising a first breath collection container for receiving a baselinebreath sample taken from the subject prior to ingestion of any of thepredetermined quantity of the uniformly labeled ¹³C-enriched glucose; asecond breath collection container for receiving a breath sample at afirst time point after ingestion of the uniformly labeled ¹³C-enrichedglucose and a third breath collection container for receiving a breathsample at a second time point after ingestion of the uniformly labeled¹³C-enriched glucose.
 14. A diagnostic kit for the determination ofglucose metabolism in a subject comprising a predetermined quantity ofuniformly labeled ³C-enriched glucose; a breath collection container forreceiving a baseline breath sample taken from the subject prior toingestion of any of the predetermined quantity of the uniformly labeled³C-enriched glucose; and at least two breath collection containers forreceiving a breath sample at predetermined time points after ingestionof the uniformly labeled ¹³C-enriched glucose.
 15. A diagnostic kitaccording to claim 14, said diagnostic kit further comprising a set ofinstructions wherein the instructions direct the subject to collect afirst breath sample in the breath collection container, ingest theuniformly labeled ¹³C-enriched glucose and collect at least two breathsamples at the predetermined time points after ingestion of theuniformly labeled ¹³C-enriched glucose in said at least two breathcollection containers.
 16. A diagnostic kit according to claim 14, saiddiagnostic kit further comprising a tube that transfers the breath ofthe subject into the breath collection containers.
 17. A diagnostic kitaccording to claim 14, said diagnostic kit further including analysismeans for measuring ¹³CO₂ metabolized from said uniformly labeled¹³C-enriched glucose, in a breath sample, said analysis means beingselected from the group consisting of an isotope ratio mass spectroscope(IRMS), a continuous flow isotope ratio mass spectroscope (CF-IRMS) anda non-dispersive infrared spectroscopy (NDIRS).
 18. The diagnostic kitof claim 17 wherein said analysis means is CF-IRMS.
 19. The diagnostickit of claim 17, wherein said analysis means is NDIRS.
 20. A diagnostickit according to claim 14 wherein said predetermined quantity contains25 mg or less of the uniformly labeled ¹³C-enriched glucose.
 21. Adiagnostic kit according to claim 14 further comprising about 15 g ofunlabeled glucose.
 22. A diagnostic kit according to claim 14, whereinsaid predetermined quantity comprises about 25 mg of the uniformlylabeled ¹³C-enriched glucose.
 23. A diagnostic kit for the determinationof glucose metabolism in a subject comprising a predetermined quantityof uniformly labeled ¹³C-enriched glucose; a first breath collectioncontainer for receiving a baseline breath sample taken from the subjectprior to ingestion of any of the predetermined quantity of the uniformlylabeled ¹³C-enriched glucose; a second breath collection container forreceiving a breath sample at a first time point after ingestion of theuniformly labeled ¹³C-enriched glucose and a third breath collectioncontainer for receiving a breath sample at a second time point afteringestion of the uniformly labeled ¹³C-enriched glucose.
 24. Adiagnostic kit according to claim 23 said diagnostic kit furthercomprising, a set of instructions wherein the instructions direct thesubject to collect a first breath sample in said first breath collectioncontainer, ingest the uniformly labeled ¹³C-enriched glucose, collect asecond breath sample at the first time point after ingestion of theuniformly labeled ¹³C-enriched glucose in the second breath collectioncontainer and collect a third breath sample at the second time pointafter ingestion of the uniformly labeled ¹³C-enriched glucose in thethird breath collection container.
 25. A diagnostic kit according toclaim 23, said diagnostic kit further comprising a tube that transfersthe breath of the subject into the breath collection containers.
 26. Adiagnostic kit according to claim 23, said diagnostic kit furtherincluding analysis means for measuring ¹³CO₂ metabolized from saiduniformly labeled ¹³C-enriched glucose, in a breath sample, saidanalysis means being selected from the group consisting of an isotoperatio mass spectroscope (IRMS), a continuous flow isotope ratio massspectroscope (CF-IRMS) and a non-dispersive infrared spectroscopy(NDIRS).
 27. The diagnostic kit of claim 26, wherein said analysis meansis CF-IRMS.
 28. The diagnostic kit of claim 26, wherein said analysismeans is NDIRS.
 29. A diagnostic kit according to claim 23, saidpredetermined quantity contains 25 mg or less of the uniformly labeled¹³C-enriched glucose.
 30. A diagnostic kit according to claim 23,further comprising about 15 g of unlabeled glucose.
 31. A diagnostic kitaccording to claim 23, wherein said predetermined quantity comprisesabout 25 mg of the uniformly labeled ¹³C-enriched glucose.
 32. A methodfor the determination of glucose metabolism in a subject comprising: a)collecting a first breath sample from said subject in a first breathcollection container; b) administering uniformly labeled ¹³C-enrichedglucose to said subject; c) collecting a second breath sample from saidsubject in a second breath collection container at a time point afteradministration of said uniformly labeled ¹³C-enriched glucose; d)collecting a third breath sample from said subject in a third breathcollection container at a second time point after administration of saiduniformly labeled ¹³C-enriched glucose; e) measuring ¹³CO₂ in each ofsaid first, second, and third breath samples; and f) comparing theamount of ¹³CO₂ in said first, second and third breath samples whereinthe difference between said amounts of ¹³CO₂ determines the glucosemetabolism in the subject.
 33. The method of claim 32 wherein steps d)and e) are repeated at least at one additional time point afteradministration of said ¹³C-enriched glucose and said comparison in stepf) compares the amount of ¹³CO₂ in all breath samples collected.
 34. Amethod of diagnosing a condition in a subject, said condition selectedfrom the group consisting of diabetes, insulin resistance, impairedglucose tolerance, impaired fasting glucose, gestational diabetes andnormal glucose metabolism, said method comprising: a) collecting a firstbreath sample from said subject in a first breath collection container;b) measuring a first blood glucose level; c) administering uniformlylabeled ¹³C-enriched glucose to said subject; d) collecting at least asecond and third breath sample from said subject in at least a secondand third breath collection container, each at a time point afteradministration of said uniformly labeled ¹³C-enriched glucose; e)measuring ¹³CO₂ in each of said first and at least said second and thirdbreath samples and comparing said measurements to obtain delta overbaseline (DOB) values; f) measuring at least a second and third bloodglucose level, each at a time point after administration of saiduniformly labeled ¹³C-enriched glucose; g) plotting said first and atleast said second and third blood glucose levels over time to define ablood glucose test curve; h) plotting said DOB values over time todefine a breath test curve; i) measuring the areas between said breathtest curve and said blood glucose test curve; and k) comparing saidareas between said breath test curve and said blood glucose test curvewith such areas measured from patients having a condition selected fromthe group consisting of diabetes, insulin resistance, impaired glucosetolerance, impaired fasting glucose, gestational diabetes and normalglucose metabolism to diagnose said conditions in said subject.
 35. Themethod of claim 34, wherein said condition is diabetes.
 36. The methodof claim 34, wherein said condition is insulin resistance.
 37. Themethod of claim 34, wherein said condition is impaired glucosetolerance.
 38. The method of claim 34, wherein said condition isimpaired fasting glucose.
 39. The method of claim 34, wherein saidcondition is gestational diabetes.
 40. A diagnostic kit for thediagnosis of a condition in a subject, according to the method of claim34, the diagnostic kit comprising: a predetermined quantity of uniformlylabeled ¹³C-enriched glucose; a breath collection container; and a bloodsampling device.
 41. A diagnostic kit according to claim 40, thediagnostic kit comprising a first breath collection container forreceiving a baseline breath sample taken from the subject prior toingestion of any of the predetermined quantity of the uniformly labeled¹³C-enriched glucose; and a second breath collection container forreceiving a breath sample after the uniformly labeled ¹³C-enrichedglucose is ingested.
 42. A diagnostic kit for the determination ofglucose metabolism in a subject by comparing blood glucose levels withbreath levels of ¹³C-enriched CO₂, said diagnostic kit comprising apredetermined quantity of uniformly labeled ¹³C-enriched glucose; ablood sampling device; a breath collection container for receiving abaseline breath sample taken from the subject prior to ingestion of anyof the predetermined quantity of the uniformly labeled ¹³C-enrichedglucose; and at least two breath collection containers for receiving abreath sample at predetermined time points after ingestion of theuniformly labeled ¹³C-enriched glucose.